Muscle wasting and negative nitrogen balance are debilitating features of many disease states including diabetes, chronic renal failure and cancer. We have recently identified a new gene, atrogin-1, which is expressed specifically in skeletal muscle and is strongly induced when muscle atrophies in various animal models of these diseases. The atrogin-1 protein contains an F-box, suggesting that it may act as a ubiquitin-protein ligase that catalyzes the ubiquitination and degradation of key proteins, leading to muscle wasting. This proposal is an outgrowth of studies which were initiated in Dr. Alfred Goldberg's laboratory, where Dr. Lecker was a postdoctoral fellow, and will now be continued mainly in the independent laboratory of Dr. Lecker. The functions and importance of this novel protein will be clarified by utilizing a combination of genetic and biochemical approaches. Muscle cells in culture will be engineered to inducibly express atrogin-1, so that its effects on muscle growth, differentiation and atrophy can be studied. The mouse atrogin-1 gene will be disrupted to generate a knockout strain where we can test whether mice lacking atrogin-1 can develop muscle atrophy. In an effort to understand how atrogin-1 might promote muscle protein breakdown, we will attempt to identify its targets and cofactors by the yeast two-hybrid approach and biochemical isolation of atrogin-1-associated proteins. Finally, we will develop in vitro assays to measure the ability of atrogin-1 to conjugate ubiquitin to proteins. Elucidating the functions of atrogin-1 will not only help characterize the mechanisms and physiological regulation of muscle protein breakdown but may also allow the development of pharmacological inhibitors that could combat muscle wasting conditions and its associated morbidity (e.g. the uremia in renal failure).